Experimental allergic encephalomyelitis (EAE), a demyelinating disease of the central nervous system, is widely used as an animal model for multiple sclerosis. We have focused our studies on the role of cytokine networks, bridging the innate and acquired immune systems, in the pathogenesis of EAE: 1. Interleukin-(IL)-12 was shown to be essential for the generation of the autoreactive Th1 cells that induce EAE, both in the presence and absence of interferon (IFNg). Although IL-12 deficient (-/-) mice were completely resistant to disease, IFNg (-/-) mice showed a markedly enhanced susceptibility to disease. Treatment of mice with anti-IFNg (-/-) resulted in exacerbation of disease, and treatment of mice with IFNg (-/-), early in disease, induced a moderate reduction in disease severity. Collectively, these observations demonstrate that IFNg exerts a protective effect in Th1 mediated autoimmunity. To determine the mechanism of protection, various parameters were analyzed in cells from IFNg -/- mice as well as in Th1 cell lines and clones stimulated in the presence of anti- IFNg. PCR analysis indicated that the absence of IFNg did not alter the expression of any known cytokines. However, stimulation of cells from IFNg -/- mice as well as T cell clones lines from normal mice in the absence of IFNg, resulted in a marked upregulation of expression , at both the RNA and protein level, of the T cell derived chemokine, TCA3. In addition, stimulation of cells from myelin basic protein (MBP)- immunized IFNg -/- mice with MBP and IFNg resulted in a marked downregulation of TCA3 mRNA expression. These studies demonstrate that upregulation of TCA3 as a result of diminished production of IFNg by antigen- specific T cells may result in migration of increased numbers of neutrophils and macrophages into the target organ thus facilitating exacerbation of disease. Taken together, these studies demonstrate that complex cytokine/chemokine networks regulate the pathogenesis of organ-specific autoimmunity. 2. We have shown that a novel IL-12/IL-10 immunoregulatory circuit controls susceptibility to autoimmune disease. During the course of studies which demonstrated that treatment of mice with anti-IL-12 suppressed their ability to produce IFNg and transfer disease, we observed that CD4+ T cells from the anti-IL-12 treated donors produced large quantities of IL-10, but not IL-4, in an antigen independent manner. Neutralization of IL-10 reversed the protection from disease afforded by treating mice with anti-IL- 12. Transfer of IL-10 producing T cells from anti-IL-12 treated unimmunized donors significantly suppressed development of EAE in actively immunized recipients. Furthermore, IL-10 -/- mice, but not IL-4 -/- mice, exhibited heightened disease susceptibility. 3. We have also critically examined the regulation of IL-12 receptor (R) expression on autoreactive T cells. MBP-specific T cells from SJL mice, but not B10.S mice, acquire an encephalitogenic Th1 phenotype following activation with antigen in vitro. The defective response of B10.S T cells can be corrected by exposure of the cells to pharmacologic concentrations of IL-12 during in vitro stimulation. This result suggested that MBP-specific T cells from B10.S mice may fail to induce IL-12 production or fail to express IL-12 receptors. To distinguish between these possibilities, B10.S and SJL mice were co-immunized with an MBP peptide and an influenza peptide (FLU). While in vitro stimulation of SJL cells with either MBP or FLU resulted in induction of IL-12Rb2 chain mRNA, cells from B10.S mice upregulated the IL-12Rb2 chain mRNA only in response to FLU. This antigen-specific defect appeared to result from the failure of induction of the CD40L on the MBP-specific T cells from B10.S mice. As CD40L/CD40 interactions play a critical role in induction of IL-12 production by macrophages and dendritic cells, these data suggest that a positive feedback loop exists between IL-12 production and IL-12R expression. The failure to generate IL-12 production early in the response to MBP in B10.S mice may be responsible for the resistance of B10.S mice to EAE. 1. We have extended the studies on the protective effect of IFNg to other models of autoimmune disease. Collagen induced arthritis (CIA) is an animal model of rheumatoid arthritis that is induced in susceptible strains of mice by immunization with heterologous type II collagen emulsified in adjuvant. CIA is considered to be a disease mediated by Th1 lymphocytes as T cells which produce IFNg are thought to play a pathogenic role in the induction of joint pathology as well as functioning as helper cells for the generation of pathogenic complement fixing IgG2a anti-collagen antibodies. The MHC plays a major role in susceptibility to CIA, as only mice of the H-2u or H-2s MHC haplotypes are susceptible. As in EAE, disruption of the gene for either IFNg or its receptor rendered normally resistant mice highly susceptible. Susceptibility was highly dependent on the presence of IL-12 as in vivo neutralization of IL-12 protected mice from disease. Surprisingly, neutralization of IL-4 either by disruption of the gene in the host animal or by administration of anti-IL-4 also significantly reduced the onset and severity of CIA. In contrast, resistant animals with disruptions of the IL- 10 gene remained highly susceptible. Thus, the protective effects of IFNg in the induction of autoimmune disease appear to be manifest in several pathologically distinct animal models. 5. As most of the animal models of organ-specific autoimmunity involve Th1 lymphocytes, we have performed an in depth analysis of the requirements for T cell receptor signalling and for costimulatory signals in the activation of Th1 cell differentiation. We have examined the effects of a set of well defined altered peptide ligands (APL) of MBP Ac 1-11 on the activation of resting CD4+ T cells from mice which express a transgenic TCR specific for this peptide. Weak agonist APL were poor inducers of all aspects of the activation of both the responder T cells and the APC. during inflammation.